Cell migration and tumor cell invasion are modulated by integrin-mediated interactions between the extracellular matrix (ECM) and the actin cytoskeleton. Dynamic regulation of adhesions and reorganization of the actin cytoskeleton are required for cell migration and are critical determinants of the invasive properties of human tumors. In previous funding periods we established that regulated intracellular proteolysis by the calcium-dependent protease calpain2 is important for cell migration by targeting specific substrates to regulate the dynamic turnover of adhesions. We now propose to investigate how calpains and associated proteins including FAK and PCH family members modulate the temporal and spatial activation of Src kinases and specific substrate proteolysis to affect adhesion dynamics during invasive cell migration. Invasive cells frequently contain two types of cell matrix adhesions known as focal adhesions and podosome-type adhesions or invadopodia. Src kinases are critical for regulating the balance between the formation of focal adhesions and the generation of matrix-degrading invadopodia, found in invasive cancer cells. We have shown that FAK and calpain2 regulate the temporal and spatial activation of c-Src and mediate a balance between focal adhesion and invadopodia formation. The hypothesis that guides this research is that calpain2 associates in complexes with FAK, Src and PCH family members, that temporally and spatially modulate c-Src activity and substrate proteolysis to affect the formation and turnover of focal adhesions (FA) and invadopodia that mediate invasive cell migration. We are now uniquely positioned to make rapid progress with the following Specific Aims: 1. Investigate how FAK/calpain2/Src complex regulates the temporal and spatial dynamics of focal adhesions and invadopodia to affect cancer cell invasion. 2. Test the hypothesis that proteolysis of specific effector substrates by calpain2 regulates adhesion dynamics and cancer cell invasion. 3. Test the hypothesis that PCH proteins, PSTPIP1 and PACSIN2, associate with calpain2 and regulate the balance between focal adhesions and podosome-type adhesions to affect invasive cell migration. The research proposed in this grant will not only elucidate the molecular mechanisms that regulate adhesion dynamics during cell migration, but will also likely provide insight into the pathogenesis of invasive cancer cell migration and the development of new therapeutics for control of disease states such as tumor invasion and metastasis.